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Sanger Sequencing
Overview Sanger sequencing is used to directly sequence DNA, which in other words, mean determining the individual nucleotides in a DNA sequence. It’s one of the earliest methods of DNA sequencing and was widely used for 25 years until it was replaced by “Next Generation” sequencing methods, which allow large-scale and automated genomic analyses. Another name for Sanger Sequencing is dideoxy sequencing and chain termination based. The practicality of Sanger sequencing made it the standard for DNA sequencing. The advantage to Sanger sequencing over new methods is its accuracy (99.99% accuracy rate). The sequence is much more accurate compared to "Next Generation" sequencing methods such as PacBio (98% accuracy rate). Usage Sanger sequencing is useful for research purposes in microbiology and molecular genetics in order to carefully analyze certain pieces of DNA. Although there are faster and more efficient sequencing technologies, Sanger Sequencing is still frequently used for small-scale projects. This is because larger DNA sequences usually are more efficiently sequenced using newer technology. Sanger sequencing is also used for longer (500-1000 nucleotide) sequences. DNA sequencing in general allows us to thoroughly analyze DNA because it gives us the fundamental information of DNA, the nucleotide sequences. Knowing the nucleotide sequence allows scientists to compare homologous genes between different species, identify mutations, and locate certain regulatory and gene sequences. Origins and Development Due to the demand for DNA sequencing methods in the early 1970s to study organisms and their functions, two methods were independently developed. The first was by a team of Americans lead by Maxam and Gilbert who developed a “chemical cleavage protocol.” The second was by a team of Englishmen lead by Frederick Sanger who designed a method similar to the DNA replication process. The 1980 Nobel Prize was shared by the English and American teams, but Sanger’s DNA sequencing method became the standard due to its reliability and speed. Sanger found out that ddNTPs or dideoxynucleotides, which lack a 3’ hydroxyl group, can terminate elongation in a growing oligonucleotide. Due to the lack of a 3’ end, ddNTPs prevent new deoxynucleotides (dNTPs, a single DNA unit) from becoming part of the new strand. ddNTPs are the same as dNTPs, which are bases normally found in DNA strand, except that they contain a hydrogen group on the 3’ end instead of a hydroxyl group. They prevent formation of a phosphodiester bond between one dNTP and the next. Running a mixture of primer, template, DNA polymerase, dNTPs, and corresponding ddNTPs in a gel shows the size distributions of dNTPs in the newly synthesized DNA strand. This is because the gel separates the fragments by size and the pattern produced from the gel can be analyzed. Since the sequence of nucleotides in the newly synthesized DNA strand is complementary to the original template DNA strand, scientists can deduce the sequence of the original strand. Method Sanger sequencing requires single-stranded DNA as the template as well as a mixture of DNA primer, DNA template, DNA polymerase, dNTPs, and corresponding ddNTPs. 1) Prior to sequencing, double stranded DNA must be heated so that they can be denatured into single stranded DNA. 2) A specially designed primer is annealed or added to one of the template strands. This primer is designed to anneal to the DNA sequence of interest. In order to visualize the final product on the gel, this primer should be labeled with a radioactive or fluorescent tag. 3) The solution of DNA with primer annealed is separated into 4 different tubes named G, A, T, and C respectively. 4) Reagents are added to each of the sample tubes. Each tube contains all four dNTPs and DNA polymerase. What separates them are the ddNTPs. ddGTP is added to the G tube, ddATP is added to the A tube, ddTTP is added to the T tube, and ddCTP is added to the C tube. Each ddNTP corresponds to a specific DNA base. 5) The mixtures in each tube are run on a gel. The longer DNA chains travel shorter distances and the shorter DNA chains travel longer distances. Examples of Use DNA sequencing has numerous uses in research. One use is to figure out the flanking sequences of a piece of DNA before making many copies of it through polymerase chain reaction (PCR). Another usage is detecting restriction sites in plasmids, which is important information used to clone a foreign gene and place it inside the plasmid. References 1. Wikipedia article: Sanger Sequencing. Date accessed: Dec 6, 2014. 2. Davidson College – Sanger Sequencing. Date accessed: Dec 6, 2014. 3. Canfield - Sanger Method. Date accessed: Dec 6, 2014. 4. Journal of Molecular Biology. Volume 94, Issue 3. 25 May 1975. Pages 441–446. 5. John Burke, UVM Professor.